1. Field of the Invention
This invention relates to fluorescent microbeads used to align fluorescent microscopes and flow cytometers, and in particular, relates to highly uniformly sized fluorescent microbeads which fluoresce over a wide range of excitation and emission wavelengths.
2. Description of the Related Art
Flow cytometers are used to analyze biological cells and particles present in a fluid sample by intersecting a thin stream of the fluid by an illumination source, usually a laser beam. The resulting forward and right angle scattered and fluorescent light is analyzed with photomultiplier tubes (PMTs). The fluorescent channels of a flow cytometer, designated by F11, F12, F13 etc., are each set with barrier filters to detect a selected specific dye while filtering out signals from dyes that fluoresce at other wavelengths.
Fluorescence instruments such as flow cytometers and fluorescence microscopes require alignment of their optical components for optimal performance. This alignment involves obtaining the most intense fluorescence signal from the instrument. More specifically in the case of flow cytometers, alignment requires obtaining the tightest grouping of events (lowest percent coefficient of variation, % CV) on the two dimensional dot plots of the fluorescence channels FL1 and FL2 as shown by comparison of the dot plots of FIG. 1 (an unaligned instrument) and FIG. 2 (an aligned instrument). This is accomplished by adjusting and focusing the optical and electrical components of the flow cytometer including the laser, lenses, mirrors, barrier filters and PMTs, so that scatter and fluorescence signals have the lowest CV.
Various excitation sources may be employed by fluorescence instruments as described by H. M. Shapiro in his book Practical Flow Cytometry, published by A. R. Liss, 1985. These excitation sources include: mercury arc lamps which have strong emission lines at 313, 334, 365, 405, 436, 546, and 577 nm; various lasers obtainable from an argon laser with excitation lines at 457, 465, 472, 476, 488, 496, 501 and 515 nm; from a helium-cadmium laser at 325 and 441 nm; and from a krypton laser at 337, 350, 406, 413, 422,520, 530, 568, 647, 676, 752, and 79 nm. Moreover, dye pumped laser can be tuned to any specific excitation wavelength across the spectrum from the UV to IR.
Usually each configuration of a fluorescence instrument requires a specific fluorescent microbead with which to align it. Instruments using a UV excitation would require a microbead which contains a dye that excites with UV radiation, whereas an instrument that uses a helium-neon laser with an excitation line at 633 nm will need a microbead that excites with red radiation.
During the past ten years, the technology required to synthesize highly uniform microbeads in the size range of 2-20 u in diameter has been developed, both in space (for example, U.S. Pat. No. 4,247,434) and on earth (for example, U.S. Pat. Nos. 4,247,434; 4,336,173; and 4,157,323). These patents, and all other patents, applications and publications cited herein, are hereby incorporated by reference.
The technology for the use of fluorescence dyes, which have very specific and limited ranges of excitation (absorption) and emission, has also been developed and improved for incorporation of the fluorescent dyes within the body and on the surface of the microbeads. In co-pending application Ser. No. 07/465,792, microbeads multiply-labeled with specific dyes were used for adjustment of a flow cytometer in multiple fluorescent channels of the flow cytometer, for subsequent measurement of a selected sample comprising cells or particles labeled with fluorescent dyes. Thus, these previous microbeads may be used to adjust multiple channels of a flow cytometer, with each microbead having the specific dyes associated with it to match a particular sample.
It is therefore an object of this invention to provide a means of aligning flow cytometers and fluorescence microscopes for use with a wide range of wavelengths, employing microbeads which fluoresce over a wide range of excitation and emission wavelengths.
It is a further object of this invention to provide a population of microbeads which fluoresce over a wide range of excitation and emission wavelengths.
Other objects and advantages will be more fully apparent from the following disclosure and appended claims.